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Vol. 18, Issue 9, 3601-3606, September 2007
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Department of Cell Biology and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06032-1507
Submitted December 9, 2006;
Revised June 19, 2007;
Accepted June 20, 2007
Monitoring Editor: Ted Salmon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Karsenti and Vernos, 2001; McIntosh et al., 2002; Scholey et al., 2003; Gadde and Heald, 2004; Wadsworth and Khodjakov, 2004).
A special role in the formation and maintenance of the mitotic spindle belongs to the minus-end–directed members of the kinesin-14 subfamily. Unlike conventional kinesin, kinesin-14 family members have motor domains at the carboxy terminus (for review, see Ovechkina and Wordeman, 2003). The best studied kinesin-14 members are Drosophila Ncd (Komma et al., 1991; Matthies et al., 1996) and Saccharomyces cerevisiae Kar3 (Meluh and Rose, 1990). These MT motors, and their mammalian (Matuliene et al., 1999; Mountain et al., 1999; Zhu et al., 2005) and plant (Vanstraelen et al., 2006) homologues, play essential roles in mitosis and meiosis. Their activities are important for the formation of the mitotic spindle poles (Goshima and Vale, 2003; Goshima et al., 2005; Morales-Mulia and Scholey, 2005; Zhu et al., 2005) and for regulation of the distance between the poles in the mitotic spindle (Saunders and Hoyt, 1992; Saunders et al., 1997; Sharp et al., 1999, 2000a; Troxell et al., 2001). Loss of a kinesin-14 function may cause different mitotic and meiotic spindle defects even within the same organism. For example, in Drosophila S2 cells and female meiosis, loss of Ncd by RNA interference (RNAi) or null mutants causes disorganization of spindle poles (Theurkauf and Hawley, 1992; Goshima et al., 2005; Morales-Mulia and Scholey, 2005), whereas in embryos, it causes changes in spindle length and a decrease in the persistence of steady-state structures (Sharp et al., 1999; Brust-Mascher and Scholey, 2002).
Evidence suggests that kinesins-14 act by driving the sliding of parallel or antiparallel MT bundles in different areas of the spindle (for review, see Sharp et al., 2000b,c; McIntosh et al., 2002; Gadde and Heald, 2004). These motors have two MT binding sites, an ATP-dependent site in the motor domain and an ATP-independent site in the tail, and they have been shown to induce MT bundling (McDonald et al., 1990; Chandra et al., 1993; Karabay and Walker, 1999; Matuliene et al., 1999). Recent small interfering RNA (siRNA) studies demonstrate that knockdown of the kinesin-14 family member Ncd results in the formation of splayed mitotic spindle poles, suggesting that the activity of this motor is required for focusing of the MTs at the pole (Goshima and Vale, 2003; Goshima et al., 2005; Morales-Mulia and Scholey, 2005). It has been found that loss of kinesin-14 function in S. cerevisiae and Drosophila leads to an increase in the spindle length and that it rescues spindle pole separation defects seen in cells lacking members of the kinesin-5 family (Saunders and Hoyt, 1992; Saunders et al., 1997; Sharp et al., 1999; Sharp et al., 2000a) and that these two motors oppose each other in in vitro motility assays (Tao et al., 2006), suggesting that the balance of their activities is required for maintaining the correct distance between the spindle poles. It is hypothesized that kinesins-14 achieve this balance by mediating antiparallel sliding of MTs emanating from the opposite poles (Sharp et al., 2000b,c; McIntosh et al., 2002; Gadde and Heald, 2004). However, despite the abundance of indirect evidence and computational models that suggest an essential role for kinesins-14 in MT sliding (Mogilner et al., 2006), such sliding activity of kinesins-14 had never been demonstrated directly before.
In this study, we developed an in vivo assay to test the hypothesis that kinesin-14 family member Ncd can induce sliding of MTs against each other in living cells. We found that green fluorescent protein (GFP)–Ncd overexpressed in cultured human fibroblasts colocalized with cytoplasmic MTs, whose localization and behavior were monitored by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies demonstrated that neighboring MTs in bundles could slide against each other with the velocities of 0.1 µm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data confirm the hypothesis that kinesin-14 family members generate sliding forces between adjacent MTs, and they constitute the first demonstration of such sliding activity of MT motors in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For the amplification of the full-size molecule or tail domain, we used the same forward primer 5'-CTC GTC GAC ATG GAA TCC CGG CTA CCG AAA CCG-3' but different reverse primers, 5'-CTC CTC GAG TTT ATC GAA ACT GCC GCT GTT GTT-3' and 5'-CTC CTC GAG TTT ATC GAA ACT GCC GCT GTT GTT-3', respectively. The gel-purified fragment was then digested with EcoR1/Sal1 and ligated into EcoR1/Sal1-digested pEGFPC2. Forward and reverse primers 5'-GAG ACC TGC AAA GAA TTC CTC TTC CAG TCG-3' and 5'-GGT GAT TTG TCG ACA GAA CCG-3' were used for amplification of the C-terminal motor domain. This fragment was then cloned into pEGFPC2 vector after restriction digestion with BamH1 and EcoR1.
An Ncd rigor-type mutant was made by introducing a point mutation T441N at the ATP-binding consensus motif. A similar kinesin mutation in the heavy chain of conventional kinesin (T93N) caused tight irreversible ATP-insensitive binding of kinesin molecules to MTs in vivo and in vitro (Nakata and Hirokawa, 1995). The forward and reverse primers used for the generation of Ncd rigor mutant were 5'CAG ACT GGT TCG GGA AAA AAT TAC ACT ATG GAC GGG GTG-3' and 5'-CAC CCC GTC CAT AGT GTA ATT TTT TCC CGA ACC AGT CTG 3', respectively. Mutant Ncd DNA was cloned into pEGFP2 expression vector similar to wild-type full-size Ncd DNA. Drosophila Kinesin-1 DNA (clone SD02406; Drosophila Genomics Resource Center, Indiana University, Bloomington, IN) was provided by Dr. William Saxton. For fusion with GFP, Kinesin-1 DNA was amplified using PCR (forward and reverse primers were 5'-GGA ATT CAT GTC CGC GGA ACG AGA G-3' and 5'-GGA TCC ACG AGT TGA CAG GAT TAA CCT G-3', respectively). The gel-purified fragment was then digested with EcoR1/BamH1 and ligated into EcoR1/BamH1-digested pEGFPN1 vector (Clontech, Mountain View, CA). GFP-tau plasmid (GFP-tau23; Samsonov et al., 2004) was a gift from Dr. Sergei Popov (University of Illinois at Chicago).
Cell Culture and Transfection
Human 356 fibroblasts were cultured in F-10 medium supplemented with 10% fetal bovine serum, and antibiotics, at 37°C. Cells were transfected with GFP-tagged full-size or truncated Ncd DNAs by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, and they were incubated for 12–16 h at 37°C to allow for the protein expression.
Microinjection and Live Cell Imaging
For fluorescent labeling of MTs, cells were pressure microinjected with Cy3-tagged bovine brain tubulin prepared by labeling with Cy3-reactive dye as described previously (Vorobjev et al., 2001) and used at a needle concentration of 8–10 mg/ml. For fluorescence speckle microscopy (Waterman-Storer et al., 1998), the needle concentration of tubulin was reduced to 0.3–0.5 mg/ml. Injected cells were incubated for at least 1 h at 37°C in a CO2 incubator to allow for incorporation of labeled tubulin into MTs.
Fluorescence images of cells were acquired using a Nikon Diaphot 300 inverted microscope equipped with a Plan x100 1.25 numerical aperture objective. Images were collected with a slow-scan back-illuminated cooled charge-coupled device camera (CH350; Roper Scientific, Trenton, NJ) driven by MetaMorph Imaging software (Molecular Devices, Sunnyvale, CA). In some experiments, fluorescence images were acquired using a spinning disk confocal microscope (PerkinElmer Life and Analytical Sciences, Boston, MA). To reduce photobleaching and photodamage, before image acquisition cells were treated with the oxygen-depleting agent Oxyrase (Oxyrase, Mansfield, OH). During image acquisition the temperature was maintained at 37°C using the Delta T controlled culture dish system (Biopthechs, Butler, PA).
Fluorescence Redistribution after Photobleaching Assay
For photobleaching experiments, cells with Cy3-labeled MTs were locally irradiated with a laser beam focused through the objective lens. Our past work indicated that illumination leaves MTs intact but that it results in the bleaching of fluorochrome in the illuminated zone (Rodionov et al., 1994). For photobleaching, the beam of a 1-W argon ion laser (Laser Physics, West Jordan, UT) was channeled into the epi-illumination system of a Nikon Diaphot 300 inverted microscope (Nikon, Tokyo, Japan). A cylindrical lens was positioned to focus the laser beam through a 100x, 1.25 aperture objective lens into an 
3-µm-wide bar in the specimen plane. The laser was operated at 514 nm and 100 mW. Some photobleaching experiments involved illuminating MTs with an argon ion laser beam by using a custom-made illumination system attached to PerkinElmer spinning disk confocal microscope, which produced 0.5–1 µm wide bar-shaped zones on MTs. Images of bleached MTs were captured with 16- or 12-bit digital cameras. Quantification of the photobleaching data was performed using MetaMorph Image acquisition and analysis software. The bleached regions were delineated with rectangles, and average fluorescence intensity values of the pixels in delineated areas were computed. After background subtraction, the data were expressed as a percentage of the average fluorescence intensity recovered after photobleaching. The 100% value was taken as the fluorescence intensity in the same region immediately before the bleach. Data were plotted an analyzed using Origin scientific graphing and analysis software (OriginLab, Northampton, MA). Quantification of the maximum rate of MT movement involved approximation of the slopes of fluorescence recovery curves using Origin software.
Immunostaining of MTs
For immunostaining with tubulin antibody, cells were fixed with methanol for 5 min at –20°C, rehydrated in phosphate-buffered saline (PBS) containing 1% bovine serum albumin, and successively incubated with rabbit affinity-purified antibody against tyrosilated tubulin (a gift from Dr. Gregg Gundersen, Columbia University) and goat anti-rabbit antibody conjugated with tetramethylrhodamine B isothiocyanate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Images of immunostained MTs were acquired using a charge-coupled device camera as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Chandra et al., 1993) that indicate that Ncd causes the formation of MT bundles.
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) and local photobleaching of tubulin fluorescence with a laser microbeam. To suppress MT dynamic instability and its contribution to MT movement, we conducted these observations in the presence of the MT-stabilizing drug Taxol under conditions that dramatically reduced the dynamic behavior of free MT ends as seen by time-lapse imaging (Supplemental Movie 1). Playback of the image sequences indicated that fluorescent speckles (Supplemental Movie 3) and bleached zones on MTs (Figure 4 and Supplemental Movie 4) moved in the cytoplasm in the presence of Ncd. Such movement has never been observed in control cells (data not shown). Because MT movement continued in the presence of Taxol, we conclude that MT movement cannot be explained by forces generated by MT dynamics. Instead, our data suggest that the observed MT movement was produced via sliding forces generated by MT-bound Ncd molecules.
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; Walker et al., 1990; deCastro et al., 1999; Tao et al., 2006). These results strongly suggest that MT movement seen in GFP–Ncd-overexpressing cells is indeed generated by the motor activity of Ncd.
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). Therefore, mutant Ncd molecules are expected to have MT-bundling activity but to lack the ability to support MT sliding against each other. Indeed, overexpression of GFP-NcdT441N in fibroblasts caused changes in MT organization similar to that seen in cells overexpressing wild-type GFP–Ncd (Figure 5). However, looping of microtubules has been never observed in the rigor mutant-transfected cells (Supplemental Movie 7), and photobleaching experiments indicated that in GFP–NcdT441N-overexpressing cells, MTs did not show movement above that observed in control untransfected cells (Supplemental Table 1 and Figure 5, A and B). These results suggest the NcdT441N mutant, unlike wild-type Ncd, was unable to induce MT sliding.
To confirm that MT sliding in cells overexpressing Ncd is explained by an inherent ability of this MT motor to concurrently bind two neighboring MTs via the binding sites located in the N-terminal tail and the C-terminal motor domains, and to rule out the possibility that sliding of MTs is a nonspecific consequence of their bundling, we performed several control experiments. We examined the organization and behavior of MTs in cells overexpressing GFP-tagged N-terminal tail domain (amino acids 1–204) or C-terminal motor domain (amino acids 327–700) of Ncd, or Drosophila kinesin-1 fused to GFP. Although the C terminus of Ncd and kinesin-1 do have motor activity, and can induce MT movement, none of these proteins has two MT binding sites; thus, they are not able to bundle MTs. We also overexpressed in cells GFP-tau fusion protein (GFP-tau23; Samsonov et al., 2004), which has a high MT-bundling activity, but lacks a motor domain and therefore cannot move MTs against each other. Fluorescence microscopy analysis of MTs in the transfected cells showed that, as expected, overexpression of tau-GFP, but not other proteins, induced the formation of MT bundles (Supplemental Movies 5 and 6 and Supplemental Figure 1). Importantly, playback of time sequences of Cy3-labeled MTs indicated that neither bundled MTs in GFP-tau–overexpressing cells, nor single MTs in cells overexpressing GFP–Ncd1–204, GFP–Ncd327–700, or GFP–kinesin-1 moved significantly, and that MT looping characteristic for cells transfected with full-size Ncd has never occurred (Supplemental Movies 5 and 6; data not shown). We conclude that MT movement seen in GFP–Ncd-overexpresssing cells is specific to Ncd; therefore, the ability to slide MTs against each other is a fundamental property of this MT motor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Walker et al., 1990; Foster and Gilbert, 2000). Our study is the first demonstration of the motor activity of Ncd in living cells. In this work, we developed a new in vivo assay that provides the ability to examine the motility properties of MT motors in a cellular context.
It is thought that Ncd exerts its functions by bundling and sliding of MTs against each other (McDonald et al., 1990; Chandra et al., 1993; Sharp et al., 2000c). This MT bundling activity is explained by the simultaneous attachment to two neighboring MTs via its ATP-dependent motor domain and ATP-independent tail located on the opposite sides of the molecule (McDonald et al., 1990; Chandra et al., 1993; Karabay and Walker, 1999). In the presence of ATP such attachment would result in relative sliding of the neighboring MTs against each other. It has been previously demonstrated in in vitro assays that Ncd causes the formation of MT bundles; however, sliding of MTs in Ncd-induced bundles has never been seen in in vivo experiments. Our in vivo assay allowed us for the first time to observe the Ncd-dependent sliding of MTs against each other.
MT sliding seen in our experiments results in a remarkable pattern of MT behavior that involves the formation of dynamic MT loops with continuously changing curvature. Our results indicate that the formation and expansion of MT loops do not require MT dynamics, are specific for Ncd-overexpressing cells, depend on the Ncd motor activity, and involve both MT binding domains located on opposite ends of Ncd molecule. Based on these observations, we propose that the looping behavior of MTs in our assay is caused by the fact that in addition to being bundled by Ncd these MTs are also anchored on the centrosome, which restricts MT movement, resulting in the bending and buckling of the MTs during Ncd-dependent MT-MT sliding (Figure 6, left). In dividing cells, MT sliding activity generated by Ncd and other members of the kinesin-14 family is thought to be involved in two aspects of mitotic and meiotic spindle morphogenesis. For one aspect, kinesin-14 family members control mitotic spindle length by generating pulling forces on the overlapping antiparallel MTs in the spindle midzone that oppose the pushing forces produced by members of the kinesin-5 family (Saunders and Hoyt, 1992; Sharp et al., 1999, 2000a,b; Kapitein et al., 2005; Tao et al., 2006) (Figure 6, right). In addition, kinesins-14 are involved in the formation of the spindle poles themselves, presumably via minus-end–directed transport of MTs nucleated by noncentrosomal mechanisms in the cytoplasm (Goshima et al., 2005; Morales-Mulia and Scholey, 2005) (Figure 6, right). In our study, we demonstrate for the first time that kinesins-14 are indeed involved in MT–MT sliding in vivo; this result supports the hypothesis about the mitotic roles of kinesin-14 members and it provides experimental evidence for both aspects of their functioning in mitosis. In vitro reconstitution experiments are underway to confirm the sliding activity of kinesin-14 family members and to reproduce the key aspects of the mitotic spindle assembly in a purified system.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Vladimir Rodionov (rodionov{at}nso.uchc.edu).
Abbreviations used: MT, microtubule.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993). Structural and functional domains of the Drosophila ncd microtubule motor protein. J. Biol. Chem 268, 9005–9013.
deCastro, M. J., Ho, C. H., and Stewart, R. J. (1999). Motility of dimeric ncd on a metal-chelating surfactant: evidence that ncd is not processive. Biochemistry 38, 5076–5081.[CrossRef][Medline]
Foster, K. A., and Gilbert, S. P. (2000). Kinetic studies of dimeric Ncd: evidence that Ncd is not processive. Biochemistry 39, 1784–1791.[CrossRef][Medline]
Gadde, S., and Heald, R. (2004). Mechanisms and molecules of the mitotic spindle. Curr. Biol 14, R797–R805.[CrossRef][Medline]
Goshima, G., Nedelec, F., and Vale, R. D. (2005). Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins. J. Cell Biol 171, 229–240.
Goshima, G., and Vale, R. D. (2003). The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Biol 162, 1003–1016.
Goshima, G., and Vale, R. D. (2005). Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells. Mol. Biol. Cell 16, 3896–3907.
Kapitein, L. C., Peterman, E. J., Kwok, B. H., Kim, J. H., Kapoor, T. M., and Schmidt, C. F. (2005). The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 435, 114–118.[CrossRef][Medline]
Karabay, A., and Walker, R. A. (1999). Identification of microtubule binding sites in the Ncd tail domain. Biochemistry 38, 1838–1849.[CrossRef][Medline]
Karsenti, E., and Vernos, I. (2001). The mitotic spindle: a self-made machine. Science 294, 543–547.
Komma, D. J., Horne, A. S., and Endow, S. A. (1991). Separation of meiotic and mitotic effects of claret non-disjunctional on chromosome segregation in Drosophila. EMBO J 10, 419–424.[Medline]
Matthies, H. J., McDonald, H. B., Goldstein, L. S., and Theurkauf, W. E. (1996). Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein. J. Cell Biol 134, 455–464.
Matuliene, J., Essner, R., Ryu, J., Hamaguchi, Y., Baas, P. W., Haraguchi, T., Hiraoka, Y., and Kuriyama, R. (1999). Function of a minus-end-directed kinesin-like motor protein in mammalian cells. J Cell Sci 112, 4041–4050.[Abstract]
McDonald, H. B., Stewart, R. J., and Goldstein, L. S. (1990). The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor. Cell 63, 1159–1165.[CrossRef][Medline]
McIntosh, J. R., Grishchuk, E. L., and West, R. R. (2002). Chromosome-microtubule interactions during mitosis. Annu. Rev. Cell Dev. Biol 18, 193–219.[CrossRef][Medline]
Meluh, P. B., and Rose, M. D. (1990). KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60, 1029–1041.[CrossRef][Medline]
Mogilner, A., Wollman, R., Civelekoglu-Scholey, G., and Scholey, J. (2006). Modeling mitosis. Trends Cell Biol 16, 88–96.[CrossRef][Medline]
Morales-Mulia, S., and Scholey, J. M. (2005). Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. Mol. Biol. Cell 16, 3176–3186.
Mountain, V., Simerly, C., Howard, L., Ando, A., Schatten, G., and Compton, D. A. (1999). The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle. J. Cell Biol 147, 351–366.
Nakata, T., and Hirokawa, N. (1995). Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport. J. Cell Biol 131, 1039–1053.
Ovechkina, Y., and Wordeman, L. (2003). Unconventional motoring: an overview of the Kin C and Kin I kinesins. Traffic 4, 367–375.[CrossRef][Medline]
Rodionov, V. I., Lim, S. S., Gelfand, V. I., and Borisy, G. G. (1994). Microtubule dynamics in fish melanophores. J. Cell Biol 126, 1455–1464.
Samsonov, A., Yu, J. Z., Rasenick, M., and Popov, S. V. (2004). Tau interaction with microtubules in vivo. J. Cell Sci 117, 6129–6141.
Saunders, W., Lengyel, V., and Hoyt, M. A. (1997). Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors. Mol. Biol. Cell 8, 1025–1033.[Abstract]
Saunders, W. S., and Hoyt, M. A. (1992). Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell 70, 451–458.[CrossRef][Medline]
Scholey, J. M., Brust-Mascher, I., and Mogilner, A. (2003). Cell division. Nature 422, 746–752.[CrossRef][Medline]
Sharp, D. J., Brown, H. M., Kwon, M., Rogers, G. C., Holland, G., and Scholey, J. M. (2000a). Functional coordination of three mitotic motors in Drosophila embryos. Mol. Biol. Cell 11, 241–253.
Sharp, D. J., Rogers, G. C., and Scholey, J. M. (2000b). Microtubule motors in mitosis. Nature 407, 41–47.[CrossRef][Medline]
Sharp, D. J., Rogers, G. C., and Scholey, J. M. (2000c). Roles of motor proteins in building microtubule-based structures: a basic principle of cellular design. Biochim. Biophys. Acta 1496, 128–141.[Medline]
Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., and Scholey, J. M. (1999). Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos. Nat. Cell Biol 1, 51–54.[CrossRef][Medline]
Tao, L., Mogilner, A., Civelekoglu-Scholey, G., Wollman, R., Evans, J., Stahlberg, H., and Scholey, J. M. (2006). A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays. Curr. Biol 16, 2293–2302.[CrossRef][Medline]
Theurkauf, W. E., and Hawley, R. S. (1992). Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J. Cell Biol 116, 1167–1180.
Troxell, C. L., Sweezy, M. A., West, R. R., Reed, K. D., Carson, B. D., Pidoux, A. L., Cande, W. Z., and McIntosh, J. R. (2001). pkl1(+)and klp2(+): two kinesins of the Kar3 subfamily in fission yeast perform different functions in both mitosis and meiosis. Mol. Biol. Cell 12, 3476–3488.
Vanstraelen, M., Inze, D., and Geelen, D. (2006). Mitosis-specific kinesins in Arabidopsis. Trends Plant Sci 11, 167–175.[CrossRef][Medline]
Vorobjev, I., Malikov, V., and Rodionov, V. (2001). Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules. Proc. Natl. Acad. Sci. USA 98, 10160–10165.
Wadsworth, P., and Khodjakov, A. (2004). E pluribus unum: towards a universal mechanism for spindle assembly. Trends Cell Biol 14, 413–419.[CrossRef][Medline]
Walker, R. A., Salmon, E. D., and Endow, S. A. (1990). The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature 347, 780–782.[CrossRef][Medline]
Waterman-Storer, C. M., Desai, A., Bulinski, J. C., and Salmon, E. D. (1998). Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr. Biol 8, 1227–1230.[CrossRef][Medline]
Zhu, C., Zhao, J., Bibikova, M., Leverson, J. D., Bossy-Wetzel, E., Fan, J. B., Abraham, R. T., and Jiang, W. (2005). Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol. Biol. Cell 16, 3187–3199.
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